Augmented classical least squares multivariate spectral analysis

ABSTRACT

A method of multivariate spectral analysis, termed augmented classical least squares (ACLS), provides an improved CLS calibration model when unmodeled sources of spectral variation are contained in a calibration sample set. The ACLS methods use information derived from component or spectral residuals during the CLS calibration to provide an improved calibration-augmented CLS model. The ACLS methods are based on CLS so that they retain the qualitative benefits of CLS, yet they have the flexibility of PLS and other hybrid techniques in that they can define a prediction model even with unmodeled sources of spectral variation that are not explicitly included in the calibration model. The unmodeled sources of spectral variation may be unknown constituents, constituents with unknown concentrations, nonlinear responses, non-uniform and correlated errors, or other sources of spectral variation that are present in the calibration sample spectra. Also, since the various ACLS methods are based on CLS, they can incorporate the new prediction-augmented CLS (PACLS) method of updating the prediction model for new sources of spectral variation contained in the prediction sample set without having to return to the calibration process. The ACLS methods can also be applied to alternating least squares models. The ACLS methods can be applied to all types of multivariate data.

RELATED INVENTIONS

This application claims the benefit of Provisional Application No.60/309,619, filed on Aug. 1, 2001, and Provisional Application No.60/311,755, filed on Aug. 9, 2001.

GOVERNMENT RIGHTS

The Government has rights to this invention pursuant to Contract No.DE-AC04-94AL85000 awarded by the U.S. Department of Energy.

FIELD OF THE INVENTION

The present invention relates generally to the field of multivariatespectral analysis and, more particularly, to a method for augmenting aclassical least squares calibration model to provide improvedpredictions of component values in unknown samples having unmodeledsources of spectral variation.

BACKGROUND OF THE INVENTION

Over the past 20 years, quantitative multivariate spectral analysis hasprimarily shifted from the explicit classical least squares (CLS) methodto the implicit principal component regression (PCR) and partial leastsquares (PLS) methods. The principle motivation for this shift is thatCLS is based on an explicit linear additive model, e.g., theBeer-Lambert law. As such, CLS has the significant limitation that itrequires the concentrations of all spectrally active components be knownand included in the calibration model before an adequate predictionmodel can be developed. On the other hand, the PCR and PLS methods canachieve excellent predictions for multivariate spectral data sets whereall of the spectrally active components have not been determined.Consequently, CLS has been relegated to solving a small set ofwell-defined linear problems with known spectrally active componentsthat adhere to the Beer-Lambert law, e.g., infrared spectra of gas-phasesamples.

Nevertheless, PCR and PLS do not have the qualitative capabilities ofCLS since they do not generate explicit estimated pure-component spectrathat can be readily interpreted. Also, they are not well suited to theadvantages of a newly developed prediction-augmented CLS (PACLS)technique as set forth in U.S. Pat. No. 6,415,233, which is incorporatedby reference herein. The PACLS algorithm provides a basis for rapidlyupdating a CLS model during prediction of component values of the targetunknown sample. PACLS adds spectral shapes (i.e., spectral intensityinformation) to the CLS estimate of the pure-component spectra duringprediction to account for spectrally active components or other spectraleffects present in the prediction samples that were not modeled duringcalibration. PACLS allows CLS models to be updated for the presence ofspectrometer drift, changes in spectrometer parts or changes in wholespectrometers, unmodeled chemical or non-chemical spectral components,as well as updating for more generalized changes such as changes instarting materials, the presence of nonlinearities, chromaticaberrations, or stray light, etc.

However, the PACLS algorithm is limited by the fact that accuratepredictions require all interfering spectral components (includingchemical and non-chemical sources of spectral variation) be explicitlyincluded during calibration or prediction. If one or more spectralinterferences were left out of the calibration, then their spectralinfluence would have to be explicitly added during prediction to correctfor their absence in the calibration model.

These limitations of the CLS model can be reduced and even eliminated bythe development of a new generalized family of algorithms, hereinafterreferred to as augmented classical least squares (ACLS). The ACLS modeluses information derived from component values and spectral residualsduring the CLS calibration to provide an improved calibration-augmentedCLS model. When the new ACLS methods are combined with the PACLSprediction algorithm, a powerful set of new multivariate capabilities isrealized such that analyses can be performed with incomplete knowledgeof interferences in the calibration and the prediction data.

The present invention further provides a generalization of ACLS methodsfor analyzing multivariate spectral data. Specific embodiments of thegeneralized ACLS methods are: spectral-residual augmented classicalleast squares (SRACLS), scores augmented classical least squares(SACLS), and concentration-residual augmented classical least squares(CRACLS) methods which all allow one to overcome the above deficiencies.The SRACLS, SACLS, and CRACLS methods are based on CLS so that theyretain the qualitative benefits of CLS, yet they have the flexibility ofPLS and other hybrid techniques in that they can define a predictionmodel even with unmodeled sources of spectral variation that are notexplicitly included in the calibration model. The unmodeled sources ofspectral variation may be unknown constituents, constituents withunknown concentrations, nonlinear responses, non-uniform and correlatederrors, or other sources of spectral variation (e.g., temperature,spectrometer drift, etc.) that are present in the calibration samplespectra.

Augmentation can also be applied to constrained alternating classicalleast squares methods (alternating between CLS calibration and CLSprediction) that are used when the reference variables, such as thepure-component spectra or component concentrations, are inadequatelyknown for standard CLS method. The ACLS methods of the present inventioncan improve the component identification with such inadequately knowndata sets.

Combining the present invention with the PACLS technique results inprediction models that are generally comparable or better in predictionability to the standard PLS models. Also, since the various ACLS methodsare based on CLS and unlike PLS, they can incorporate the PACLS featureof updating the prediction model for new sources of spectral variationwithout the need for time-consuming recalibration. These updatedprediction models only require spectral information while PLS requiresspectral and concentration information during recalibration. The presentinvention is not restricted to using continuous spectral information,but can also use any set of discontinuous spectral intensities that areselected in the calibration for the least squares analysis. Finally, thepresent invention generates better qualitative information about theanalytes by generating better estimates of their pure-component spectra.

SUMMARY OF THE INVENTION

A method of multivariate spectral analysis is provided that is able togenerate accurate and precise prediction models from multivariatespectral data which includes unmodeled spectrally active componentspresent in the calibration model. The method provides the improvedqualitative information of CLS methods as well as the quantitativeprediction ability of the implicit multivariate calibration methods byaugmenting the calibration model with a measure of the residualresulting from unmodeled components.

The present method of multivariate spectral analysis is most useful whenan underlying calibration model includes unmodeled sources of spectralvariation. In particular, reference variables (e.g., sample spectra andcomponent values) are obtained for a set of calibration samples. Anestimate is obtained for at least one reference variable for the set ofcalibration samples. Thereafter, a residual is obtained between the atleast one reference variable and its corresponding estimated variable. Ameasure of the residual between the estimated and reference variable isused to augment its corresponding reference variable. Using theaugmented reference variable, a value of at least one component in a setof unknown samples is predicted. Augmentation can be repeated until allsources of spectral variation are accounted for in the calibrationsamples. The iterative process allows the development of a CLS-typecalibration model comparable in its quantitative prediction ability toimplicit multivariate calibration methods, even when unmodeledspectrally active components are present in the calibration samplespectra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts pictorially Eq. (16).

FIG. 2 depicts pictorially Eq. (17).

FIG. 3A is a flow diagram of the SRACLS embodiment of the presentinvention. FIG. 3B is a flow diagram of the SACLS embodiment of thepresent invention.

FIG. 4 is a flow diagram of the CRACLS embodiment of the presentinvention.

FIG. 5 depicts four loading vectors (solid lines) estimated fromsimulated spectra using the CRACLS calibration that includes urea andwater concentrations and two concentration residual augmentations. Thepure-component spectra (dotted lines) for water, glucose, urea, andethanol are placed close to the corresponding loading vector (LV) forshape comparison.

FIG. 6 depicts mean-centered simulated spectra showing high frequencynoise.

FIG. 7 depicts shape comparisons of the first weight loading vector(WLV) of a PLS glucose calibration and the first loading vector of a CLScalibration including glucose and water using the simulated data withthe pure-component spectrum for glucose.

FIG. 8A depicts the mean spectrum and 8B depicts the mean-centeredspectra of the calibration spectra.

FIG. 9A depicts the difference between average repeat spectra for thecalibration and prediction data sets and 9B depicts the mean-centeredaverage repeat spectra from the prediction data set.

FIGS. 10A, 10B and 10C depict the ethanol, urea and water concentrationresiduals, respectively, vs. the reference glucose concentration fromthe CLS model applied to the simulated data when leaving out glucoseconcentrations.

FIG. 11 is a flow diagram of the ACLS method of the present invention asapplied to an alternating least squares model.

FIG. 12 shows a video image of the thin neoprene sample aged for 6 daysin air at 140° C.

FIG. 13A depicts spectra of neoprene pixels from spectral image of theneoprene sample. FIG. 13B depicts spectra of neoprene bands fromspectral image of the sample after removing a linear baseline from eachof the 6 neoprene related spectral bands.

FIG. 14 shows examples of systematic artifacts in the spectral imagescores FIG. 14A shows scores from factor 5 of an interferogram. FIG. 14Bshows scores from factor 10 of the negative log of a sample single beam.FIG. 14C shows scores from factor 2 of two ratioed backgroundsingle-beam spectra after conversion to absorbance. FIG. 14D showsscores from factor 3 from same two ratioed background single-beamspectra after conversion to absorbance.

FIG. 15 depicts mean-centered repeat spectra from the neoprene pixels inthe spectral image.

FIG. 16A shows the covariance error structure of the mean-centeredrepeat spectra. FIG. 16B shows the ideal error covariance structureassumed for standard least-squares analyses.

FIG. 17 depicts the experimental and theoretical estimates for theweighting function. The upper trace is the inverse of the square root ofthe variance component from the mean-centered repeat spectra (leftaxis). The lower trace is the average single-beam spectrum of theneoprene pixels (right axis).

FIG. 18 depicts the estimated pure-component spectra using standardnon-negativity constrained MCR.

FIG. 19 depicts the concentration maps for components 1, 2, and 3obtained from the standard non-negativity constrained MCR.

FIG. 20 depicts the estimated pure-component spectra obtained byaugmenting with the first five scores from the mean-centered repeatspectra and using non-negativity and equality constrained MCR.

FIG. 21 depicts the concentration maps for components 1, 2, and 3obtained by augmenting with the first five eigenvectors from themean-centered repeat spectra and using non-negativity and equalityconstrained MCR.

FIG. 22 depicts the estimated pure-component spectra obtained byaugmenting with the first five sets of scores and eigenvectors from themean-centered repeat spectra and using non-negativity and equalityconstrained MCR.

FIG. 23 depicts the concentration maps for components 1, 2, and 3obtained by augmenting with the first five sets of scores andeigenvectors from the mean-centered repeat spectra and usingnon-negativity and equality constrained MCR.

FIG. 24 depicts the differences in the concentration maps of components1, 2, and 3 from the standard MCR and MCR augmented with the first fivesets of scores and eigenvectors from the mean-centered repeat imagespectra of the neoprene pixels.

DETAILED DESCRIPTION OF THE INVENTION

To better understand the present invention, the following introductorydiscussion is provided. The notation in the equations below usesupper-case bold letters for matrices, lower-case bold letters forvectors, and italicized letters for scalars. We use the {circumflex over( )} to indicate estimated values, ^(T) to denote a transposed matrix,⁻¹ for matrix inversion; ⁺ for the pseudoinverse of a matrix, and {tildeover ( )} for augmented matrices. The use of the pseudoinverse canresult in improved numerical precision from a variety of methods, e.g.singular value decomposition. Vectors are column vectors with rowvectors indicated by the transpose of the vector.

As used herein, the term multivariate spectral data includes all typesof multivariate data (i.e., data related to each sample or object iscomposed of responses from two or more variables). As used herein,spectral data to be used in the multivariate spectral analysis areconsidered to be any form of data where there are responses at two ormore measured variables (e.g., wavelength, time, retention time,frequency, etc.) due to one or more perturbation sources (e.g.,electromagnetic radiation, vibration, temperature, mechanical impulses,etc.). Therefore, the present invention can be applied to conventionalspectroscopic methods that include, but are not limited to, infrared,near-infrared, visible, ultraviolet, X-ray, gamma-ray, Raman, massspectroscopy, ion-mobility mass spectroscopy, Auger, fluorescence,phosphorescence, ESCA, far-infrared, microwave, x-ray fluorescence, NMR,energy loss spectroscopy, EDAX, ESR, and multi- and hyper-spectralimaging spectroscopy. These spectroscopic methods can be used inabsorption, transmission, reflection, or emission modes. In addition,the methods of the present invention can be applied to other forms ofmultivariate data that include, but are not limited to, seismic data,chromatographic data, thermal gravimetric analysis, image data, andresponses from multiple sensors. In particular, the methods of thepresent invention can be used to provide quantitative analysis of imagedata of the type provided with recently available commercialhyper-spectral imaging spectrometers. The methods can also be applied tothe analysis of multispectral and hyperspectral image data obtained fromremote sensors, such as satellite or airborne hyperspectral imagingsensors.

Classical Least Squares

The basic CLS method can be described by the following relationship:

A=CK+E _(A)  (1)

where A is a n×p matrix of measured spectra from n calibration samplesat p frequencies, C is a n×m matrix of reference component values foreach of the m spectrally active components, K is a m×p matrix of in situpure-component spectra scaled to unit concentration and pathlength foreach component m, and E_(A) is a n×p matrix of spectral residuals (i.e.,model errors, if the model is not linear or if K does not include allthe pure-component spectra, and spectral noise) for each frequency p.Although a component can typically be a chemical species and a componentvalue can typically be a concentration of the chemical species, the termcomponent as used herein generally applies to any source of spectralvariation, be it chemical, physical or otherwise. Examples of sources ofspectral variation that can be modeled include the effect of theindividual chemical species, chemical interactions, or any changescaused by a wide variety of physical parameters which can inducespectral variations, such as temperature variations, spectrometer drift,humidity changes, and sample insertions. Note that the physical propertyof temperature can have a considerable quantifiable effect on the samplespectra as can be seen from samples containing an aqueous solvent. TheCLS calibration step requires determining the least-squares estimate forpure component spectra from a set of calibration samples with knowncomponent values. These estimated pure-component spectra are given by:

{circumflex over (K)}=(C ^(T) C)⁻¹ C ^(T) A=C ⁺ A  (2)

where C is the matrix of reference component values for the spectrallyactive components in the calibration sample set and A is the matrix ofmeasured spectra for the calibration sample set.

In a CLS prediction step, the estimated pure-component spectra can thenbe used to predict the component values in an unknown set of samplesaccording to:

Ĉ=A _(P) {circumflex over (K)} ^(T)({circumflex over (K)}{circumflexover (K)} ^(T))⁻¹ =A _(P)({circumflex over (K)} ^(T))⁺  (3)

where now A_(P) is the matrix of measured spectra for the unknownsamples.

Eq. (2) provides accurate estimates of the known pure-component spectra{circumflex over (K)}, and therefore accurate predictions of the unknowncomponent values Ĉ, only if the data fit a linear model and C containsall the components that contribute to spectral variation in thecalibration sample spectra A. The accuracy of the estimatedpure-component spectra {circumflex over (K)} improves with eachadditional known reference value c_(i) added to the CLS model.Therefore, the present invention is most useful in analyzingmultivariate spectral data when there are unmodeled sources of spectralvariation in the calibration model.

Augmented Classical Least Squares

The present invention provides a family of methods for analyzingmultivariate data where there are unmodeled sources of spectralvariation in the calibration model. The family of methods can be broadlydescribed as first obtaining reference variables for a set ofcalibration samples (e.g., reference spectral data and reference valuesfor components in the set of calibration samples). An estimate isobtained for at least one of the reference variables for the set ofcalibration samples. Thereafter, a residual is obtained between the atleast one reference variable and its corresponding estimated variable.The residual between the estimated and reference variables is used toaugment its corresponding reference variable. Using the augmentedreference variable, a value of at least one component in a set ofunknown samples is predicted. Augmentation can be repeated until allsources of spectral variation are accounted for in the set ofcalibration samples. The iterative process allows the development of aCLS-type calibration model comparable in quantitative prediction abilityto implicit multivariate calibration methods even when unmodeledspectrally active components are present in the calibration samplespectra. The present invention generates an adaptable model that canachieve excellent quantitative and qualitative prediction results forsamples of unknown composition that contain unmodeled sources ofspectral variation.

In the spectral-residual-augmented classical least squares (SRACLS)embodiment of the present invention, a spectral residual matrix E_(A)can be determined from a CLS estimate of the pure-component spectra{circumflex over (K)}, reference values C, and calibration spectra A:

E _(A) =A−C{circumflex over (K)}  (4)

The rows of the estimated pure-component spectra {circumflex over (K)}matrix can be augmented directly with all or selected vectors from thespectral residual matrix E_(A) to correct the CLS model for unmodeled,spectrally active components. The augmented matrix {tilde over({circumflex over (K)})} can then be used in an augmented CLS predictionto obtain predicted component values {tilde over ({circumflex over(C)})} for a prediction set of unknown samples:

{tilde over ({circumflex over (C)})}=A _(P) {tilde over ({circumflexover (K)})}({tilde over ({circumflex over (K)})}{tilde over ({circumflexover (K)})} ^(T))⁻¹ =A _(P) {tilde over ({circumflex over (K)})} ⁺  (5)

where now A_(P) represents the spectral matrix of the unknown samples tobe predicted.

For an incompletely specified and/or nonlinear model, the spectralresidual E_(A) includes a set of correlated, non-uniform errors that canbe further decomposed into a sum of correlated errors, due to unmodeledspectral components, nonlinearities, or system-related correlatederrors; and uncorrelated random errors representing system noise and/orspectral variation that are not relevant to prediction.

Factor analysis methods can be applied to the spectral residual E_(A) toseparate the sources of these errors. Thus, the spectral residual E_(A)can be described as:

E _(A) =TP+E  (6)

where T and P are the set of n×r scores and r×p loading vectors,respectively, obtained from the factor analysis of the spectralresiduals E_(A), and E is the set of n×p random errors and spectralvariations not useful for prediction. The dimension r is the rank of thespectral residual matrix E_(A) or the number of factors that arerequired for optimal prediction. Therefore, Eq. (1) can be re-written asfollows:

A=CK+TP+E  (7)

In one embodiment of the present invention, Eq. (6) can be factoranalyzed by any factor analysis method. Common methods include principalcomponent analysis (PCA), partial least squares (PLS), or principalcomponent regression (PCR). If PLS or PCR is used, then concentrationerrors (to be discussed later) will be included in the calibrationprocess, as described in U.S. Pat. No. 6,341,257, which is incorporatedherein by reference. The factor analysis can use either an orthogonalfactor analysis method or any non-orthogonal factor analysis method.

Using only the spectral residuals in the factor analysis, the scores, T,or the loading vectors, P, can be used to augment the CLS calibrationmodel. If PCA is used as the factor analysis method applied to thecalibration spectral residuals E_(A), then the eigenvectors, P (i.e.,the rows of P), can augment the rows of the {circumflex over (K)} matrixusing the SRACLS model to improve the prediction ability of the CLSmodel. The augmented CLS prediction for component values {tilde over({circumflex over (C)})} can then be obtained according to Eq. (5).Martens and Naes (Multivariate Calibration; John Wiley & Sons:Chichester 1989) suggested this approach for removing the effects ofunmodeled components. However, Martens and Naes did not use theaugmented pure-component spectra {tilde over ({circumflex over (K)})} incombination with a PACLS algorithm for prediction. When combined withPACLS (wherein spectral shapes obtained from data or information thatare independent of the calibration data set can be used to update thespectral responses), the SRACLS model can be updated in a PACLSprediction step for the presence of unmodeled changes in the responsesof the prediction samples.

The scores T can also be used to correct the CLS calibration model forunknown spectral components. In this case, the columns of T can augmentthe column dimension of the component values C matrix to form anaugmented component value matrix {tilde over (C)}. Re-calibrating withthe augmented CLS method as in Eq. (8) results in an augmented {tildeover ({circumflex over (K)})} matrix that allows the augmented CLS modelto have better predictive properties than the original CLS model:

{tilde over ({circumflex over (K)})}=({tilde over (C)} ^(T) {tilde over(C)})⁻¹ {tilde over (C)} ^(T) A≈{tilde over (C)} ⁺ A  (8)

When augmentation makes use of scores, this embodiment is referred to asscores-augmented classical least squares (SACLS). Indeed, one can alsosimply augment the component values C matrix with one or more columns ofrandom numbers. In general, any set of vectors of the correct dimensionfor T can be used to improve the CLS model as long as they each containsome independent information relative to themselves or the original Cmatrix, i.e., they cannot be collinear with each other or C.

With both SRACLS and SACLS, the spectral residuals E_(A) can be obtainedfrom the full calibration model or from cross-validated calibrations.The optimal number of augmentations in either SRACLS of SACLS can beselected based on an F-test of the concentration residuals (usingcross-validated concentration residuals) according to methods describedby Haaland and Thomas, Anal. Chem. 60, 1193 (1988) and Haaland andThomas, Anal. Chem. 60, 1202 (1988), both of which are incorporatedherein by reference.

Another ACLS method involves the use of component value residuals E_(C)(e.g., concentration residuals) to augment the component matrix C. Whenaugmentation makes use of concentration residuals , the method isreferred to as concentration-residual-augmented classical least squares(CRACLS). In general, each estimated pure-component spectrum willconsist of the pure-component spectrum at unit reference value for thecorresponding known reference component in C plus linear combinations ofthe unmodeled pure-component spectra, i.e. $\begin{matrix}{{\hat{k}}_{j} = {k_{j} + {\sum\limits_{i = 0}^{m^{u}}\quad {b_{i}k_{i}^{u}}}}} & (9)\end{matrix}$

where {circumflex over (k)}_(j) represents the estimated pure-componentspectrum for the j^(th) component and b_(i) are the linear scale factorsfor the m^(u) unmodeled pure-component spectra k_(i) ^(u). In the CRACLSmethod, these estimated pure-component spectra {circumflex over (k)}_(j)can then be used to estimate the reference component values Ĉ asfollows:

Ĉ=A{circumflex over (K)} ^(T)({circumflex over (K)}{circumflex over (K)}^(T))⁻¹ =A({circumflex over (K)} ^(T))⁺  (10)

An n×m component residuals matrix, E_(C), then is given by:

E _(C) =Ĉ−C  (11)

The component residuals E_(C) can be obtained from the entire set ofcalibration data after the CLS steps in Eqs. (9) and (10).Alternatively, the component residuals can be obtained fromcross-validated predictions on calibration samples rotated out duringthe cross-validation procedure or from prediction on a set of validationsamples that are not part of the calibration set.

To account for baseline variations, {circumflex over (K)} can beaugmented with explicit baseline functions, i.e., vectors representingthe potential variations in baselines in the spectra. These baselinefunctions can include an offset, general polynomials, orthogonalLegendre polynomials, or any expected functional form of the baselines.A row is added to {circumflex over (K)} for each additional order in thebaseline function added. Using the PACLS technique, estimated ormeasured spectral shapes for other spectrally active components can beadded to {circumflex over (K)} as well. A corresponding column must beadded to Ĉ for each added row in {circumflex over (K)} before solvingEq. (10). If rows have been added to {circumflex over (K)}, then E_(C)is computed using only the estimated reference values corresponding tothe original reference values in C.

To consider unmodeled sources of spectral variation, Eq. (1) can berewritten:

A=CK+C _(u) K _(u) +E  (12)

where C_(u) is an n×m^(u) matrix that represents the unknown componentvalues for each of the m^(u) unmodeled pure-component spectra in K_(u),and E represents the error remaining after removing m^(u) unmodeledlinear spectral effects. Even if E_(A) in Eq. (1) is the result ofnonlinear factors, the nonlinearities can be estimated by a linearapproximation in C_(u)K_(u), so even in those cases, E can beconsiderably smaller then E_(A).

The matrix, K_(u), can be decomposed into the sum of two terms. One partof K_(u) can be written as the projection of K_(u) onto the spacespanned by K, (i.e., P(K)K_(u)=DK), where D is a m^(u)×m matrix andm^(u) is the number of rows in K_(u) and m is the number of rows in K.Another part of the decomposition of K_(u) is orthogonal to K, denotedby G. The decomposition of K_(u) is then:

K _(u) =DK+G  (13)

Substituting Eq. (13) into Eq. (12) and gathering terms yields:

A=(C+C _(u) D)K+C _(u) G+E  (14)

The estimated component values Ĉ in Eq. (10) are then approximatelyequal to (C+C_(u)D). Consequently from Eq. (11), we have

E _(C) ≅C _(u) D  (15)

Although D is not known, Eq. (15) still shows that each column, e_(c),of E_(C) approximates a linear combination of the unknown componentvalues unless K_(u) is orthogonal to K, (i.e., D=0, where 0 is the nullmatrix). However, if D=0, the unmodeled components will not contaminatethe estimated component values Ĉ so they can be ignored withoutaffecting the predicted component values.

In practice, D=0 will generally occur only if the spectral components donot overlap in the spectral region being analyzed. For calibration ofthe known components, the magnitudes for unit component value of thepure-component spectra for the unmodeled components are not required,since only the relative component values are needed to generate thecorrect net-analyte signals (NAS) for each of the known components. Byincluding linear combinations of the unmodeled component values asadditional columns in C and solving Eq. (16), the resulting additionalpure-component spectra {tilde over ({circumflex over (K)})} will belinear combinations of the spectra of the unmodeled pure-components,K_(u). When enough linear combinations are added to cover all sources ofspectral variation beyond the noise, the NAS for each of the knowncomponents in {tilde over ({circumflex over (K)})} will be correct.

Selecting one vector of the component residuals, e_(c), and augmentingthe original C matrix with the selected residual vector e_(c) as a newcolumn creates the augmented n×(m+1) matrix, {tilde over (C)}. If thereare more than one unmodeled spectrally active components, only one ofthe component residual vectors e_(c) is used in the augmentation, sincethe residuals for the other unmodeled components will contain redundantinformation. Criteria for selecting which component residual vectore_(c) to use will be discussed in more detail below.

Using {tilde over (C)}, the augmented, (m+1)×p, pure-component spectramatrix {tilde over ({circumflex over (K)})} can be computed accordingto:

{tilde over ({circumflex over (K)})}=({tilde over (C)} ^(T) {tilde over(C)})⁻¹ {tilde over (C)} ^(T) A={tilde over (C)} ⁺ A  (16)

This step of the CRACLS method is illustrated in FIG. 1 where thecomponent residual vector e_(c) used to augment the reference componentvalue matrix C are given as the e_(i)′s, and the additional estimatedpure-component spectrum in Eq. (16), is represented by the dotted line.

By iteration, additional augmentations can be used to remove additionalunmodeled sources of spectral variation. The new augmented estimatedpure-component spectra {tilde over ({circumflex over (K)})} from Eq.(16) can then be used to again estimate reference component values{tilde over ({circumflex over (C)})} for the calibration samplesaccording to:

{tilde over ({circumflex over (C)})}=A{tilde over ({circumflex over(K)})}K ^(T)({tilde over ({circumflex over (K)})}{tilde over({circumflex over (K)})} ^(T))⁻¹ =A({tilde over ({circumflex over (K)})}^(T))⁺  (17)

where here A are the calibration spectra. This estimation step is shownin FIG. 2 where the c_(ei)′s represent the estimated component valuesfor the additional pure-component spectrum (i.e., the dotted line).Since the additional spectrum is a linear combination of unspecifiedmagnitude of the unmodeled pure-component spectra, the c_(ei)′s in{tilde over ({circumflex over (C)})} may not provide any usefulquantitative information. A new n×m component residual matrix, E_(C)′,then is given by:

E _(C) ′=Ĉ′−C  (18)

where Ĉ′ is an updated estimated component value matrix consisting onlyof the estimated component values in {tilde over ({circumflex over(C)})} corresponding to the known reference values in C.

The steps delineated by Eqs. (11, 16-17) remove one linear combinationof the unmodeled spectral variations from the calibration model. Thesesteps can be repeated again using one row from E_(C)′ to further augmentthe component value matrix. To mitigate all the additional sources ofspectral variation (i.e., reduce the residual from E_(A) in Eq. 1 to Ein Eq. 14), these steps can be repeated for each of the independentsources of spectral variation present in the calibration data. In otherwords, the component residual vectors e_(c) can span the space definedby the unmodeled spectrally active components. In all augmented CLSmethods, the optimal number of augmentations can be determined using thesame method for PLS factor selection that has been previously publishedfor PLS and PCR. See, e.g., Haaland and Thomas, Anal. Chem. 60, 1193(1988) and Haaland and Thomas, Anal. Chem. 60, 1202 (1988). In thisfactor or vector selection method, a maximum number of augmentationsgreater than the optimum number of augmentations is selected, and anACLS model is determined for each level of augmentation up to thismaximum number using cross-validation techniques. The optimal number ofaugmentations is then determined based on the F-test usingcross-validated component residuals (from the full calibration model orfrom cross-validated calibration models) for each level of augmentation,similar to the procedure described by Haaland and Thomas. The componentresiduals E_(C) in the CRACLS method can be generated from either thefull models or the cross-validated models.

Using the fully augmented {tilde over (C)}, augmented pure-componentspectra {tilde over ({circumflex over (K)})} can be calculated accordingto Eq. (16). This augmented pure-component spectral matrix {tilde over({circumflex over (K)})} can then be used to predict component values{tilde over ({circumflex over (C)})} in the unknown samples according toEq. (5).

Because any of the ACLS models represent a CLS-type model, they are wellsuited to take advantage of the newly developed PACLS technique. ThePACLS algorithm further augments the augmented pure-component spectra{tilde over ({circumflex over (K)})} prior to prediction with spectralinformation representing unmodeled sources of spectral variation presentin the unknown sample spectra to be predicted. Basically, {tilde over({circumflex over (K)})} from the calibration model can be furtheraugmented prior to the prediction step in Eq. (5) with morepure-component spectra that encompass spectrally active components inthe prediction data set that were not present in the calibration model.The advantage of PACLS is that the calibration model can be updatedquickly during prediction to account for new sources of spectralvariation. Recomputing the calibration model using the original and theadditional spectra, as required by the prior art, would takeconsiderably more time.

The spectra added during prediction should account for all sources ofspectral variation introduced during the collection of the predictiondata that were not accounted for in the calibration model, as describedin the previously referenced U.S. Pat. No. 6,415,233. If these sourcesof spectral variation can be attributed to a known component, a samplecould be doped with the identified component to determine a spectralshape that can be added to the prediction. However, often the newsources of spectral variation (such as caused by instrument drift orsample insertion effects) cannot be so easily identified. Frequently, arepeat sample can be used to provide the required spectral informationto augment the model for prediction. By repeatedly measuring a stablesample during calibration and prediction spectral measurements, spectralshapes for these new sources of spectral variation can be captured. Thedifferences between repeated spectral measurements obtained during bothcalibration and prediction or an eigenvector analysis of thosedifferences can provide the spectral information required foraugmentation of the prediction model.

Note that, unlike PLS, the spectral variation can be incorporated usingthe PACLS method without knowing the values of various components. Whenusing the repeat sample spectra, the assumption is that the spectra canbe used to provide a reasonable estimate of the error covariance matrix,i.e., that the unmodeled sources of spectral variation affect the repeatsample spectra and the prediction sample spectra in the same way. Thisassumption is valid if the spectra are similar across all samples, whichis the case for the dilute aqueous solutions considered herein. However,if the range of component values causes large variations in the samplespectra, it may be necessary to use a subset of repeat samples toadequately capture the impact of the unknown sources of spectralvariation.

The following discussion of the SRACLS method of the present inventionis made with reference to FIG. 3A. The present invention comprises twosteps: calibration (steps 10-90) and prediction (steps 100-130).

Initially, a set of calibration samples containing a plurality ofspectrally active components is obtained at 10. A set of calibrationspectra A are obtained for some or all of the samples in the calibrationsample set at 20. Additionally, a set of reference component values C isconstructed for at least one of the spectrally active components in thecalibration sample at 30. Such component values C typically provide ameasure of the concentration of one or more known spectrally activecomponent within the set of calibration samples. However, those skilledin the art will appreciate that such component values can representother factors representative of the effects of both chemical andphysical phenomenon that are spectrally active (i.e., can alter thespectral data such as temperature). With the calibration spectra A andcomponent values C, Eq. (2) can be used at 40 to obtain CLS calibrationmodel estimates of the pure-component spectra {circumflex over (K)}.

At step 45, baseline variations as well as estimated or measuredspectral shapes representing other unmodeled components can be added tothe estimated pure-component spectra {circumflex over (K)}.

At step 60, spectral residuals E_(A) representative of the differencesbetween the calibration spectra A and the estimated calibration spectracan be obtained according to Eq. (4) using the estimated pure-componentspectra {circumflex over (K)}. The spectral residual matrix E_(A) can bedecomposed by factor analysis methods to separate the sources of thespectral residuals.

The spectral residuals can be decomposed per Eq. (6). All or selectedspectral residual vectors from E_(A) or P can be used to augment therows of the estimated pure-component spectra {circumflex over (K)} at 90(i.e. to obtain augmented pure-component spectra {tilde over({circumflex over (K)})}) at least once or until all unmodeled,spectrally active components in the calibration sample set have beenaccounted for. Any of a variety of methods to using spectral orcomponent value residuals, variation or other properties can be used toselect the vectors for augmentation.

In FIG. 3B is shown the SACLS method of the present invention. Here, thescores T can be used to correct the CLS model for unknown components. Inthis case, the columns of T can be added to C at 80 to augment thecolumn dimension of C to form an augmented component value matrix {tildeover (C)}. Re-calibrating with this augmented CLS method as in Eq. (8)results in an augmented {tilde over ({circumflex over (K)})} matrix atstep 90 that allows the augmented CLS calibration model to have betterpredictive properties than the original CLS model. All or selected scorevectors can be added to C and the augmented CLS model recalibrated atleast once or until all unmodeled, spectrally active components havebeen accounted for in the calibration set in the augmentedpure-component matrix {tilde over ({circumflex over (K)})}. Any of avariety of methods can be used to select the scores for augmentation.

In FIGS. 3A and 3B, the augmented CLS prediction for component values{tilde over ({circumflex over (C)})} of the unknown samples thenproceeds in the prediction steps 100-130. The SRACLS or SACLS estimateof the pure-component spectra {tilde over ({circumflex over (K)})} canbe further augmented with spectral data using the PACLS technique(wherein spectral shapes obtained from data or information that areindependent of the calibration sample set can be used to update thespectral responses) and/or with baseline variations at step 100.Thereafter, at step 110, a predicted component value matrix {tilde over({circumflex over (C)})} for a prediction set of unknown samples 130 canbe obtained according to Eq. (5), where now A_(p) in step 120 is thematrix of the measured spectra for the unknown samples.

The following discussion of the CRACLS embodiment of the presentinvention is made with reference to FIG. 4. This embodiment alsocomprises two steps: calibration (steps 10-90) and prediction (steps100-130).

Initially, a set of calibration samples containing a plurality ofspectrally active components is obtained at 10. A set of calibrationspectra A is obtained for some or all of the samples in the calibrationsample set at 20. Additionally, a set of reference component values C isconstructed for at least one of the spectrally active components in thecalibration samples at 30. With the calibration spectra A and componentvalues C, Eq. (2) can be used at 40 to obtain a CLS calibration modelthat comprises estimates of the pure-component spectra {circumflex over(K)}.

At step 45, baseline variations as well as estimated or measuredspectral shapes representing unmodeled components can be added to theestimated pure-component spectra {circumflex over (K)}.

By extension at 50, an estimate of the component values Ĉ can beobtained with Eq. (10). At step 70, a component residual matrix E_(c)representative of the differences between the estimated component valuesĈ and the reference component values C can be obtained according to Eq.(11). The component residual matrix E_(c) can be decomposed into acomponent residual vector e_(c) for each component included in thecalibration.

Each component residual vector e_(c) is representative of at least oneunmodeled spectrally active component. A selected component residualvector e_(c) can augment the original component values matrix C at 80 toform an augmented component values matrix {tilde over (C)}.Alternatively, a first estimate of the residual vector can simply bedrawn from random numbers. The augmented component value matrix {tildeover (C)} can be used to obtain an augmented estimate of thepure-component spectra {tilde over ({circumflex over (K)})} at step 90according to Eq. (16). Using the singly augmented pure-component spectra{tilde over ({circumflex over (K)})}, the CRACLS method can transitionto prediction.

Alternatively, in another embodiment with iterations indicated by thecircular arrow, steps 85-70-80-90 can be repeated to further augment thepure-component spectra {tilde over ({circumflex over (K)})}. Withiteration, an augmented component value matrix {tilde over ({circumflexover (C)})} is calculated according to Eq. (17), from which an updatedestimated component value matrix Ĉ′ can be obtained at step 85. Thisupdated component value matrix Ĉ′ can then be used to calculate a newcomponent residual matrix E_(C)′ according to Eq. (18) at step 70. Aselected component residual vector e_(c)′ can then be used to furtheraugment the augmented component values matrix {tilde over (C)} at step80, from which a new augmented pure-component spectral matrix {tildeover ({circumflex over (K)})} can be obtained at step 90. This iterationcan be repeated until all unmodeled, spectrally active components in thecalibration sample set have been accounted for.

After the desired number of augmentations, the augmented CLS predictionfor component values {tilde over ({circumflex over (C)})} of the unknownsamples then proceeds in the prediction steps 100-130. The estimate ofthe pure-component spectra {tilde over ({circumflex over (K)})} can befurther augmented with spectral shapes representing spectrally activecomponents in the prediction samples that where not modeled incalibration steps using the PACLS technique and/or with baselinevariations at step 100. Thereafter, at step 110, a predicted componentvalue matrix {tilde over ({circumflex over (C)})} for a prediction setof unknown samples 130 can be obtained according to Eq. (5), where nowA_(P) in step 120 is the matrix of the measured spectra for the unknownsamples.

EXAMPLE 1 CRACLS Method Applied to the Analysis of Non-imaging Data

The CRACLS method of the present invention is demonstrated with bothsimulated and real data derived from a system of dilute aqueoussolutions containing glucose, ethanol, and urea. The simulated datademonstrate the effectiveness of the CRACLS algorithm and help toelucidate the principles behind the method. Using experimental data, theprediction abilities of CRACLS and PLS are compared duringcross-validated calibration. In combination with PACLS, the CRACLSpredictions are comparable to PLS for the prediction of the glucose,ethanol, and urea components for validation samples collected whensignificant instrument drift was also present.

The experimental samples consisted of a series of dilute aqueoussolutions of glucose, ethanol, and urea each independently varied overthe concentration range of 0-500 mg/dL. The samples were prepared byweight and volume in a pseudo D-optimal design that allowed each of thethree analytes to be varied separately at 9 levels over theconcentration range. The aqueous solvent was obtained from a singlesource of buffered saline solution. A detailed error analysis indicatedthat the samples were made to an accuracy of better than 1 mg/dL. Thecalibration data set consisted of 27 samples plus a repeat sample takenfrom the center of the design, i.e., the repeat sample containedapproximately 250 mg/dL each of glucose, ethanol, and urea. The set ofsamples used for prediction (i.e., the validation samples) included thesame repeat sample and 27 new samples from a design similar to thecalibration set. The validation samples spanned the same 0 to 500 mg/dLconcentration range for each of the analytes as in the calibration set.No sample from the new 27-sample prediction set had the same compositionas any sample in the calibration set. Five of the prediction sampleswere removed from consideration when they were determined to be outliersamples contaminated by the epoxy used to seal the lids on the cuvettes.

The samples were sealed with a magnetic stirring bar in 10-mm pathlengthcuvettes. A temperature controller propelled the magnetic stirrer whileholding the samples at a temperature constant to 0.05° C. (±1σ). Thesamples were placed in the temperature controller and held in the beamof the spectrometer for 4 minutes with stirring to allow the sampletemperature to equilibrate. The near-infrared spectra of the sampleswere obtained on a Nicolet Model 750 Fourier transform infrared (FT-IR)spectrometer. The spectrometer employed a 75 W tungsten-halogen lamp,quartz beam splitter, and liquid-N₂-cooled InSb detector. Spectra at 16cm⁻¹ resolution were obtained after averaging the interferograms over a2-minute period.

The run order of the calibration sample set was randomized. The spectrumof the repeat sample held at 32° C. was obtained after each group of twocalibration or prediction samples. The spectra for the predictionsamples were obtained approximately one month after the calibrationspectra were obtained. Purge variation was introduced during theprediction data set to induce additional short-term instrument drift andto accentuate the difficulty in maintaining the calibration. Sampletemperature was varied in random order in 2° C. steps over the range of30 to 34° C. for both the calibration and prediction samples. Abackground of the empty sample holder was obtained after each sample.Transmittance spectra of the calibration and validation sample sets wereobtained by ratioing each single-beam sample spectrum to either itscorresponding background or to the average of the background for theday. The spectra were then converted to absorbance. Since the bestcalibration and prediction results were obtained with the spectraobtained using the averaged daily background, only the results obtainedfrom these spectra are described herein.

Simulated Data

To demonstrate the effectiveness of the present invention, a simulatedspectral data set was constructed using pure-component spectra derivedfrom experimental dilute aqueous data using standard CLS methods. Whilethese simulated spectra do not precisely match the true pure-componentspectra (primarily because of significant baseline variations resultingfrom spectrometer drift during the collection of the calibrationspectra), they can be used to demonstrate the capabilities of thepresent invention. The simulated spectral data were constructed fromthese pure-component spectra and a series of assumed concentrations togenerate two different sets of 25 samples, one for calibration and theother for validation, containing 0-500 mg/dL each of glucose, ethanol,and urea and 98000-100000 mg/dL of water. The sum of all the componentsfro each sample was constrained to 100000. In addition, normallydistributed random spectral noise was added to the absorbance spectra ata level of 0.3% of the maximum spectral absorbance intensity. This levelof noise amounted to approximately 5% of the absorbance caused by theminor components, which is slightly more noise than we observed in themeasured spectral data.

The spectral data were analyzed in the spectral region from 7500 to11,000 cm⁻¹ using the CLS, PLS and CRACLS models. The calibration modelsfor each technique were used for prediction of the validation samplespectra.

The pure-component spectra K of the analytes and the water solvent usedto generate the simulated spectral data are shown in FIG. 5. The glucosepure-component spectrum is similar to that of a modified water spectrumsince glucose has no noticeable vibrational features of its own in thisregion of the spectrum. Rather, the CLS-estimated glucose spectrum isdominated by the interaction of glucose with the water solvent.Consequently, building a multivariate spectral model to estimate glucoseconcentration in the samples is more difficult than for the otheranalytes. On the other hand, both ethanol and urea have distinctivespectral features (e.g., at 8500 cm⁻¹ and 9900 cm⁻¹, respectively) sothey are more easily quantified.

Since water is the dominant component, the spectra of the simulatedsamples closely resemble that of water. However, if we remove theaverage spectrum, the spectral variation due to the analytes of eachsimulated sample is apparent as shown in FIG. 6. Most of the spectralvariation shown is due to differences in the concentrations of theanalytes between samples, which are accentuated by the baselinevariations present in these pure-component spectra. The high-frequencycomponent superimposed on the spectra is the added noise.

The effect of augmentation on the prediction ability of the CRACLS modelwas examined for the simulation samples. If only two of the fourcomponents, glucose and ethanol, are used to build a CLS model based onthe simulated data, the cross-validated standard errors of prediction(CVSEP) for glucose and ethanol are 234 mg and 93 mg, respectively. Thatis, there is almost no prediction ability for glucose. However, if aCRACLS model is built, augmenting the calibration model with theconcentration residuals twice, CVSEPs of 17 mg for glucose and 3 mg forethanol are achieved, demonstrating that the present invention methodhas clear prediction ability for both components. If only onecomponent's concentrations is included in the CRACLS calibration model,either glucose or ethanol, and the model is augmented three times, weget essentially the same CVSEPs are obtained as above. Adding theconcentration residuals E_(c) to the reference concentration matrix Csuccessfully removed contamination of the unmodeled components from thecalibration model for the included components.

The CRACLS loading vectors qualitatively resemble the pure-componentspectra. The estimated pure-component spectra or loading vectors (LV)that resulted by including water and urea concentrations in thecalibration and augmenting with concentration residuals twice are shownas the solid lines in FIG. 5. The vectors have been shifted slightly toshow the comparisons more clearly with the pure-component spectra (thedotted lines). Also, the loading vector for ethanol was multiplied by −1to match the orientation of the pure-component spectrum. The qualitativeinformation of the loading vectors is apparent when compared with theactual pure-component shapes shown using the dotted lines. LV1 and LV2closely resemble the pure-component spectra of the given components,urea and water, respectively. Also, even though ethanol was not includedin the calibration, LV3 resembles its pure-component spectrum. Becausethe glucose has weak vibrational bands in this region, its spectralinformation is not apparent in any of the loading vectors, even in LV4.

If the major component is left out of the CLS calibration, thequalitative information will not be as apparent. The loading vectorswill generally match the shape of the major component due to itsoverwhelming influence. The extent of this influence on the simulateddata is shown by the decomposition of the vectors in the next section.With mean centering, the loading vectors obtained from a CLS calibrationwithout the solvent concentrations will reveal more of the distinctivespectral features of the non-solvent components. However, for a mixturesystem where the sum of the concentrations is constrained to a constant,the CLS estimated pure-component analyte spectrum represents the netchange in the sample spectrum due to a unit change in the analyteconcentration. Consequently, the estimated pure-component spectrum willinclude negative spectral changes due to the displacement of the solventthat will contaminate the mean-centered loading vectors.

In every case, the qualitative information in the loading vectors forCLS will be equal to or better than PLS, since the first PLSweight-loading vector that retains the best qualitative information isgenerated from only one component. For CLS, and therefore for CRACLS,the qualitative information is improved as additional analyteconcentrations are included in the calibration model. To illustratethis, a PLS calibration on glucose and a CLS calibration using onlyglucose and water were computed using the simulated data. The weightloading vector (WLV) from the PLS calibration and the loading vector(LV) from the CLS calibration for glucose are given in FIG. 7 as thedashed and dotted lines, respectively. The PLS weight loading vector iscontaminated by the displacement of the water by the analyte, so thespectral shape does not match the pure-component shape (solid line) verywell. The loading vector from a CLS calibration using only glucose andmean centering has the same shape. However, by including the waterconcentration, its contamination is removed, and the CLS first loadingvector as shown in FIG. 7 provides an improved match to thepure-component spectrum. The more information included in the CLScalibration, the better the fit. If the spectrometer drift has a linearcomponent, adding time of spectrum collection to the CLS concentrationmatrix will yield better pure-component spectra. Also CLS will generatebetter pure-component spectra than PLS when the known componentconcentrations are correlated.

The CRACLS loading vectors may be decomposed to demonstrate theeffective removal of unmodeled sources of spectral variation, even ifonly the minor components are included in the calibration. Thedecomposition of the first four CRACLS loading vectors provides insightinto this effectiveness when only the minor components of glucose andethanol are included in the calibration without mean centering. Bydefining a CLS prediction model using all four true pure-componentspectra with unit concentration, shown in FIG. 5, that were used tocreate the simulated data, the contribution of each of the true spectrato the loading vectors can be quantified as shown in Table I.

TABLE I Quantity of pure-component spectra in CRACLS loading vectorsusing glucose and ethanol concentrations and two augmentations. GlucoseEthanol Urea Water mg/dL mg/dL mg/dL mg/dL LV 1 1.0 0.0   0.4   196 LV 20.0 1.0   0.5   134 LV 3 0.0 0.0 −0.1 −150 LV 4 0.0 0.0 −1.0  −48

The spectral residuals, after removing the given amount for each of thepure-component spectra, represent only random noise, indicating that allthe spectral information contained in these loading vectors was removed.As shown in the Table I, the first and second loading vectors are theonly ones that contain the glucose and ethanol pure-component spectra,respectively, corresponding to the order that their concentrations wereplaced in the concentration matrix. Note also that the pure-componentspectra are present at unit concentration so they can providequantitative information during prediction. In addition, the urea andwater spectra contaminate these first two estimated pure-componentspectra, as expected from Eq. (9).

In Table I, the amount of water far exceeds the other components, so allof the loading vector shapes are very similar to the pure waterspectrum. The last two estimated pure-component spectra are linearcombinations of only urea and water, the components left out of theconcentration matrix. Consequently, during prediction using these fourloading vectors, the resulting NAS for glucose and ethanol (i.e., thepart of each pure-component spectrum orthogonal to all other vectors) isthe same as obtained from the fully specified CLS model. Basically, thecontamination of both water and urea is removed from the prediction ofthe glucose and ethanol by the third and fourth loading vectors.Therefore, the prediction model correctly determines the glucose andethanol concentrations even though the concentrations for the other twocomponents were not explicitly included in the calibration.

Results From the Experimental Data

Now we consider the experimental data. The mean calibration spectrum andthe mean-centered calibration sample spectra are shown in FIGS. 8A and8B, respectively. As with the simulated data, the water spectralfeatures dominate the spectra before mean centering. However, most ofthe spectral variations in the mean-centered spectra shown in FIG. 8Bare not due to concentration differences, but rather are the result ofspectrometer drift and temperature variations. Due to these additionalsources of variation, even a fully specified CLS model is not able toadequately model the data. This fact is clearly demonstrated in theCVSEPs from the full CLS model of the real calibration data shown in thefirst column of Table II.

TABLE II Minor component CVSEPs for dilute aqueous solutions using CLS,PLS, and CRACLS calibration models. CRACLS CRACLS CRACLS CLS PLS^(a)(10)^(b) (15)^(b) (20)^(b) Glucose (mg/dl) 71 15 (11) 18 16 15 Ethanol(mg/dl) 23  6 (11)  6  5  5 Urea (mg/dl) 18 6 (9)  6  5  5 ^(a)Value inparentheses is the optimal number of PLS factors used in the model.^(b)Values in parentheses represent the number of augmentations used inthe CRACLS calibration.

All the chemical components and temperature were included in the model,and yet CLS still was unable to generate a precise calibration model.These results indicate why CLS is seldom used for prediction inmultivariate calibrations. Also provided in Table II are the CVSEPs forPLS, with the optimum number of factors shown in parenthesis, and forCRACLS, using 10, 15 and 20 augmentations. Although there was a gradualdecrease in the CVSEPs with more augmentations, the optimal number ofaugmentations was not apparent. Differences between the presentinvention calibration results and the PLS results for these data are notstatistically significant.

To predict the validation data, information from the repeat sample wasincluded in both the PLS and the CRACLS models. For PLS, all the spectrafrom the repeat sample taken with the prediction data set were added tothe original calibration data set along with its componentconcentrations, and the PLS model was recalibrated. The cross-validatedcalibrations were performed initially by using the standard approach ofremoving all the data for each sample during the rotations (sample-outrotation), so that all the spectra for the repeat sample were removed atonce. The number of loading vectors or factors for the PLS model wasdetermined using an F-test on the cross-validated calibrations.

For the present invention, no changes were made in the CRACLScalibration model, i.e., no information from the repeat sample was givento the original CRACLS model to compute new pure-component spectra.Instead, the sources of spectral variation from the repeat sample shownin FIGS. 9A and 9B were used (without concentration information) toaugment the pure-component matrix during prediction. The advantage ofthis PACLS augmentation is that the prediction model can be rapidlyupdated to account for new sources of spectral variation in theprediction data set. Recomputing the calibration model using theoriginal and the additional spectra from the repeat sample would takeconsiderably more computation time. The spectral difference between theaverage of the repeat spectra from the calibration and prediction datasets, shown in FIG. 9A, was added to the prediction model to capture thelong-term spectrometer drift between the calibration and predictiondays. Without the addition of this mean-difference spectrum, thepredicted values show a definite bias. In addition, all themean-centered repeat spectra for the prediction repeat sample, shown inFIG. 9B, were added to eliminate the detrimental effects of anyshort-term instrument drift during the collection of the predictionsample spectra.

By mean centering, the spectral contribution of the repeat sample'sspecific concentration is removed, leaving only the variation fromnon-chemical sources. Note the sharp features in the mean-centeredspectra at approximately 8800 cm⁻¹ and 10600 cm⁻¹ in FIG. 9B. Thesefeatures are the result of water vapor variations caused by the changesin the quality of the purge. Other less obvious spectrometer driftfeatures contribute to the spectral variation as well. The backgroundspectra are inadequate to correct for all sources of spectrometer drift.In fact, prediction results were best when an average background, ratherthan individual backgrounds, was used to obtain the absorbance spectra.

Table III shows the standard error of predictions (SEPs) for all theminor constituents in the prediction data set from various CLS, PLS andthe present invention methods using calibration models based upon thecalibration data set and the repeat sample information.

TABLE III CVSEP for PLS and SEPs for CLS, PLS, and CRACLS on theprediction data set using models containing the repeat sample spectralinformation. CRACLS/ CRACLS/ PACLS PACLS Component PLS CLS PLS-A^(a)PLS-B^(a) (10)^(b) (20)^(b) Glucose 18 287 55 (6)  21 (12) 19 19 (mg/dl)Ethanol  5  27 9 (8)  4 (13)  5  4 (mg/dl) Urea  5  42  4 (11)  4 (12) 4  4 (mg/dl) ^(a)Value in parentheses is the optimal number of PLSfactors used in the model. ^(b)Values in parentheses represent thenumber of augmentations used in the CRACLS calibration.

As a comparison standard, the first column of Table III provides theCVSEP obtained from a separate PLS calibration obtained from theprediction data. As expected, CLS without the information about theinterferents had poor prediction ability. PLS-A in Table III representsPLS recalibration with the optimal number of PLS factors selected duringthe sample-out cross-validated rotation. The results show that the modelwas inadequate, even though this method of rotating spectra out duringcross-validation has generally been recommended to avoid overfitting thedata.

Therefore, the cross-validated rotation was modified duringrecalibration so that each of the repeat spectra from the prediction setwas rotated out one at a time (spectrum-out rotation). By using thespectrum-out cross-validated rotation, a more correct number of factorsare selected, and the PLS-B model in Table III predicted well for thesedata. Cross-validated rotation removing individual spectra was preferredin this case over rotation removing all repeat spectra for a givensample since the repeat spectra were added to capture the system drift,not the component concentrations. By rotating all repeat spectra out atonce, the F-test on the cross-validated calibrations did not adequatelygauge the impact of the drift on the residuals for factor selection.While the spectra-out rotation worked better for this data set, it canlead to severe overfitting in other data sets. Adding several spectrafor the same sample can over emphasize that sample in the model, whichcould lead to overfitting. Consequently, a careful analysis on acase-by-case basis is required to determine the appropriate type ofspectral rotation during cross-validation for PLS. The proper choice ofrotation for PLS will depend on the relative effects of theconcentrations and the spectrometer drift or other sources of spectralvariations that need to be modeled.

The results for present invention models using 10 and 20 augmentationsduring the CRACLS calibration are also presented in Table III. Again, itcan be seen that the present invention compares favorably with PLS.Since the CRACLS model was developed using all components and the PLSmodel uses only one component at a time, it could be argued that thecomparison is not valid. Therefore, the results were recalibrated usingthe present invention with 20 augmentations and the concentrations fromonly one component at a time to generate three separate models. Applyingthese models to the prediction data set, the SEPs for glucose, ethanoland urea were 20, 4, and 4 mg/dL, respectively. Essentially, theseresults matched the values derived when all the components were used inthe CRACLS calibration model. Clearly, either way, the present inventionmethod effectively removed the impact of all sources of spectralvariation not represented by the given/known concentrations. Notice,however, that with CRACLS there is no problem with selecting a rotationmethod since the mean-centered repeat spectra are simply augmented tothe estimated pure-component spectra during prediction.

When there is more than one known component, the concentration residualvector to use for augmentation needs to be chosen. To do this, theimpact that the unmodeled components will have on each of theconcentration residuals needs to be considered. From Eq. (15), theconcentration residual vector is related to the projection of theunmodeled pure-component spectra, K_(u), onto the space spanned by themodeled pure-component spectra, K, i.e. the portion of the unmodeledpure spectra that overlaps with the modeled pure spectra. If one of theunmodeled and one of the modeled pure-component spectra happen to beorthogonal, the concentration residual from that modeled component willnot provide any information about the concentrations of the unmodeledcomponent. However, in practice, orthogonal pure-component spectrararely occur since orthogonality generally implies that there is nospectral overlap. Even using simulated data with pure-component spectraconfigured to be nearly orthogonal, augmentation with the concentrationresidual vector was still sufficient to produce a good predictive model.

Returning now to the simulation, to demonstrate the applicability of thevarious component residuals, a calibration model was developed using thesimulated data, leaving out only the glucose concentrations duringcalibration. The concentration residuals vs. the reference concentrationfor the three components included in the calibration are shown in FIGS.10A, 10B and 10C. The absolute value of the residuals varies from 20 to100 mg/dL depending on the analyte. However, the pattern of the sampleresiduals for each of the components is basically the same, indicatingthat the contamination from glucose contributed approximately the samerelative error to each of the samples for each of the components.Consequently, any of these component residuals will give statisticallyequivalent CRACLS models since the magnitude of the differences willonly change the scaling factor of the estimated glucose pure-componentspectrum corresponding to the augmented residual vector, C_(u), in Eq.(15). The resulting NAS of the known components will be essentiallyidentical regardless of which residual vector is selected.

Another choice for CRACLS is selection of the number of augmentations touse in the model. When using PLS, it is important to avoid using toomany factors since overfitting the concentration data will degrade theprediction model. The excessive PLS factors have concentration residualsassociated with them that can degrade the concentration predictions ifthey are included in the model. Finding the correct number ofaugmentations in CRACLS, however, may be less critical than choosing theoptimal number of factors for PLS. Each additional loading vectorgenerated by the concentration residual augmentation in CRACLS resultsin a reduction of the NASs for the analytes of interest. After the majorunmodeled sources of spectral variation have been removed, the additionof more concentration residual vectors will generate estimatedpure-component spectra that represent primarily random noise. Sincerandom spectral noise is nearly orthogonal to the pure-component spectraof the known analytes, the impact on the NAS will be minimal. Theinsensitivity of CRACLS to the number of augmentations was demonstratedin the example above using real data. As shown in Tables II and III, themodels generated using 10 or 20 augmentations gave equivalent resultswith no evidence of overfitting. A sufficient number of residual vectorsneed to be added to capture all of the unmodeled sources of spectralvariation, but adding more residual vectors did not degrade the results.For these data, the CVSEPs for CRACLS dramatically decreased withincreasing augmentations as real sources of spectral variation weremodeled, and then the CVSEPs just gradually continued to decrease. Theapparent insensitivity of CRACLS to the number of augmentations is anadvantage over PLS since, at times, it is difficult to determine theoptimal number of factors for PLS. The F-test for PLS can also be usedon the cross-validated calibrations to select the number ofaugmentations in CRACLS. This F-test often results in selecting themaximum or near the maximum number of augmentations computed since theCVSEPs often just gradually decrease with each augmentation.

An important observation is that the PACLS-augmented prediction modelsare mathematically identical whether the estimated of measured spectralshapes representing additional unmodeled sources of spectral variationin the prediction data set are added to {circumflex over (K)} of Eq.(10) during the cross validation in the calibration phase or to {tildeover ({circumflex over (K)})} before performing prediction. In addition,equivalent prediction ability was achieved with CRACLS by using all thecomponents simultaneously or by using the concentrations one componentat a time. However, to enhance the sensitivity to outlier detection andto achieve better qualitative information in the estimatedpure-component spectra, it is recommended to include all known referenceconcentrations in the cross-validated calibration.

EXAMPLE 2 ACLS Method Applied to the Analysis of Hyperspectral ImageData

Multivariate methods use a large number of spectral channels to takeadvantage of the signal averaging and resolving power inherent inspectral data. The multivariate nature of the spectra and the massiveamount of data in hyperspectral images allow the characterization ofsystems with low signal-to-noise ratios and with multiple componentswhose spectral features overlap. Multivariate data also provide theopportunity to correct systematic spectral artifacts and aberrationsintroduced by the imaging instrument.

The quantitative analysis of hyperspectral image data collected fromFT-IR imaging systems that employ focal plane array (FPA) detectors canbe greatly hampered by the presence of system artifacts in the spectraland image data. Methods are required to eliminate the detrimentaleffects of these systematic artifacts if they cannot be eliminatedexperimentally. The characteristics of the systematic artifacts can becaptured in the error covariance structure of the data estimated fromrepeat spectral image differences. By coupling the generalized ACLSmethod of the present invention with improved multivariate curveresolution (MCR) techniques and estimates of the error covariancestructure of the data, the detrimental effects of the systematicartifacts present in hyperspectral imaging FT-IR systems can be greatlyminimized.

MCR is a constrained alternating classical least squares method(alternating between CLS calibration and CLS prediction) used when thereference variables, such as pure-component spectra or componentconcentrations, are inadequately known for the standard CLS method. SeeR. Tauler et al., “Multivariate Curve Resolution Applied to SpectralData from Multiple Runs of an Industrial Process,” Anal. Chem. 65(15),2040 (1993) and Bro and Dejong, “A fast non-negativity-constrained leastsquares algorithm,” J. of Chemometrics 11(5), 393 (1997), both of whichare incorporated herein by reference. Rather, MCR methods useconstraints, such as non-negativity, equality, closure, monotonicconstraint, unimodality, or selectivity, to achieve a solution.Therefore, MCR seeks to identify components of a data set by usingalternating classical least squares to iteratively obtain solutions foreach set of variables that putatively constitute the data. Because ofthe adaptability of the present invention and the fact that MCR employsa series of alternating classical least squares steps, ACLS methods canbe readily added to the MCR algorithms to improve the componentidentification with such data sets.

Alternating Classical Least Squares

Conventional MCR methods start with a guess for either the K or Cmatrices, using random numbers or using other methods to arrive at areasonable first guess, such as SIMPLISMA. See W. Windig and D. A.Stephenson, Anal. Chem. 64, 2735 (1992). If the starting guess is K,then CLS prediction is performed, with the appropriate constraint(s)applied (non-negativity, equality, etc.) to narrow the range ofpotential solutions, to provide a predicted component values matrix Ĉ,according to:

Ĉ=AK ^(T)(KK ^(T))⁻¹ =A(K ^(T))⁺  (19)

where A is the matrix of measured spectra for the unknown sample.Alternating to CLS calibration, estimated pure-component spectra{circumflex over (K)} can then be obtained, again using constraints,according to:

{circumflex over (K)}=(Ĉ ^(T) Ĉ)⁻¹ Ĉ ^(T) A=Ĉ ⁺ A  (20)

The steps depicted by Eqs. (19) and (20) can be repeated, updating thepure-component spectra K in Eq. (19) for each iteration with newestimated pure-component spectra {circumflex over (K)} from Eq. (20),and testing convergence according to:

∥A−Ĉ{circumflex over (K)}∥ ²  (21)

until the set of iterations converges, minimizing the constrainedclassical least squares solution (i.e., a measure of the spectralresidual E_(A) is minimized).

Augmented Alternating Classical Least Squares

For any alternating least-squares MCR method, the calibration andprediction CLS steps can be augmented with information that it isdesired for the least-squares fit to ignore (e.g., vectorsrepresentative of known or estimated spectral components or any estimateof the error covariance of the system). An estimate of the errorcovariance can be obtained, for example, by taking repeat spectra or, inthe case of spectral imaging, repeat spectral images. Each least-squarescalibration step in MCR iteration can be augmented by the scoresobtained from the factor analysis of the error covariance matrix or eachleast-squares prediction step can be augmented by the loading vectorsfrom the same matrix, or both steps can be augmented. Additionalconstraints can be applied to the non-augmented portion only of theaugmented matrices. In this manner, quantitative analysis of spectralimages can proceed without the need for standards even in the presenceof large error covariance in the data. In general, ACLS methods will beuseful in the analysis of any type of spectral data where estimates ofthe error covariance of the data can be obtained.

In FIG. 11 is shown an augmented alternating CLS embodiment of thepresent invention. A starting guess is made of the pure-componentspectra K at step 200. This starting guess can also be a matrix ofrandom numbers. In addition, an estimate of the error covariance E_(A)of the measured spectra A is obtained. With spectral image data, thisestimate of the error covariance E_(A) can be obtained from thedifference of repeat spectral images or from shift differences obtainedfrom a single spectral image when repeat spectral images are notavailable. The estimate of the error covariance E_(A) can then be factoranalyzed into the product of T scores and P loading vectors. A preferredfactor analysis method is PCA. The guessed pure-component spectra K canbe augmented with one or more of the vectors of the P loading vectors atstep 260 to obtain augmented pure-component spectra {tilde over (K)}.The guessed pure-component spectra K can also be augmented with one ormore vectors representing spectral shapes that are representative of atleast one additional source of spectral variation according to the PACLSmethod. A first set of augmented component values {tilde over({circumflex over (C)})} can then be predicted at step 110 according to:

{tilde over ({circumflex over (C)})}=A{tilde over (K)} ^(T)({tilde over(K)}{tilde over (K)} ^(T))⁻¹ =A({tilde over (K)} ^(T))⁺  (22)

where A is the matrix of measured spectra for the unknown sample fromstep 210. Additional constraints can be applied to the non-augmentedportion of {tilde over ({circumflex over (C)})} at step 220. The firstset of augmented component values {tilde over ({circumflex over (C)})}can be augmented with selected T scores at step 230 by replacing theaugmented values in {tilde over ({circumflex over (C)})} with theappropriate scores from T to further improve the solution. An estimatedaugmented pure-component spectral matrix {tilde over ({circumflex over(K)})} can be obtained at step 90 according to: $\begin{matrix}{\hat{\overset{\sim}{K}} = {{\left( {{\hat{\overset{\sim}{C}}}^{T}\quad \hat{\overset{\sim}{C}}} \right)^{- 1}\quad {\hat{\overset{\bullet\bullet\bullet}{C}}}^{T}\quad A} = {{\hat{\overset{\sim}{C}}}^{+}\quad A}}} & (23)\end{matrix}$

Constraints can be applied to the non-augmented portion of {tilde over({circumflex over (K)})} at step 240 to further narrow the range ofpossible solutions. The first set of augmented component values C fromstep 110, or the replaced set of augmented component values {tilde over({circumflex over (C)})} from step 230, and the estimated augmentedpure-component spectra {tilde over ({circumflex over (K)})} from step 90can then be used to test for convergence at step 250 according to:

∥A−{tilde over ({circumflex over (C)})}{tilde over ({circumflex over(K)})}∥ ²  (24)

These CLS prediction and calibration steps with constraints andaugmentation can be repeated in a series of iterations indicated by thecircular arrow until the set of iterations converges, minimizing theconstrained augmented classical least squares solution. Alternatively,the initial guess can be C with the CLS calibration being performedfirst. The procedure is then similar to the above-described modified MCRmethod with only the starting point changing. These modified MCR methodscan be applied to any set of multivariate data, and the use of augmentedCLS methods is demonstrated below with spectroscopic image data.

To demonstrate the improvements possible with augmented CLS methods, theMCR analysis with augmentation were applied to a FT-IR spectral imageobtained from a sample of neoprene thermally aged in air at elevatedtemperatures containing system artifacts in the image data. With theACLS method applied to the spectral image data, augmentation with scoresimproved the pure-component spectral estimates while augmentation withthe corresponding eigenvectors improved primarily the relativeconcentration maps predicted from the image data. Augmentation with bothscores and loading vectors (eigenvectors) improved both thepure-component spectra and the concentration maps, even without therequirement for calibration standards.

Experimental

A sample of neoprene rubber was investigated. To obtain adequate signalto noise in the FT-IR image data, the neoprene sample did not containthe normal carbon filler found in many neoprene rubbers. The sample thatwas used to illustrate the methods of the present invention was obtainedfrom a 2 mm×4 mm×4 mm block of neoprene that was aged for 6 days in anair furnace held at a temperature of 140° C. The sample was then pottedin epoxy and the center of the sample was cut with a microtome to anapproximate thickness of 15 μm. However, independent estimates of thesample thickness as a function of position on the sample indicated thatthe thickness varied by a factor of greater than three across thesample.

The spectrometer used in this study was a BioRad Stingray step-scanFT-IR imaging spectrometer equipped with a mercury-cadmium-telluride(MCT) FPA detector that had 4096 (64×64) detector elements. The samplewas placed in the macro sampling compartment that provided a 4 mm×4 mmtransmission image to be obtained of the thin 2 mm×4 mm aged neoprenesample. The sample was pressed flat onto a KBr window for support.Background single-beam images were obtained from a clear region of theKBr window next to the neoprene sample. Sample single-beam transmissionimage spectra were then obtained of the neoprene sample. The spectrawere collected at a nominal spectral resolution of 16 cm⁻¹ using a steprate of 2.5 steps/sec and co-adding 64 images at each step of theinterferometer. Data collection time was approximately 5 minutes foreach spectral single-beam image. Three image spectra were obtained inrapid succession of the KBr window background before moving the sampleinto the beam. Three image spectra were then obtained from the neoprenesample without moving the sample. Two of these repeat image spectra wereused to obtain an estimate of the error covariance structure of thedata.

Analysis

The spectral data were collected with the use of the Bio-Rad Win-IR Prosoftware that came with the Stingray spectrometer. However, spectralanalyses were performed using the Matlab code for the MCR, ACLS, andSIMPLISMA algorithms.

Small numbers of spectra from each image were clear outliers and wereremoved from the analysis. These outliers generally exhibited spikes orsteps in the raw interferograms. The sample images were trimmed toremove all pixels representing just the KBr window and mixed pixels atthe extreme boundaries of the sample that contained sample and KBrwindow. Only pixels present in all single-beam images were retained forfinal analysis. The sample spectra were ratioed to backgrounds andconverted to absorbance. Large, relatively complex baseline variationswere present in these spectra. The presence of these baseline variationscomplicated attempts to extract pure-component spectra. Therefore, thespectra were preprocessed by isolating the spectral features of thesample and performing a separate linear baseline correction under eachspectral band for each spectrum in the image.

When pathlength varies over the imaged area of the sample, concentrationinformation can be confounded with pathlength changes, since pathlengthand concentration changes are generally indistinguishable. Thesepathlength variations can be largely corrected if the Beers-Lambert lawis closely followed and if a spectral region can be identified thatrepresents only pathlength variations. Since the neoprene rubber samplethickness was not uniform over the sample area, pathlength correctionsto the data were required to obtain meaningful concentration maps of thevarious components across the sample. To perform pathlength correctionswithout direct measurements of the sample thickness, a spectral bandthat was most representative of sample pathlength was identified. Thisband was selected among the six baseline-corrected bands by performing aprincipal components analysis (PCA) on each spectral band and examiningthe results carefully. Necessary conditions for a spectral band to berepresentative of sample pathlength are that the band be present in allpixels of the image, that it has a consistent spectral shape in allpixels, and that only multiplicative changes are present in the band.Spectral bands that fulfill these conditions of only a multiplicativechange will have the first PCA eigenvector represent nearly all thevariance, and the first eigenvector will have the same shape as theaverage spectrum of the selected band. Of the six bands identified inthe neoprene sample, the 1440 cm⁻¹ band most nearly followed thesecriteria and was, therefore, selected as representing sample pathlength.

Once an appropriate spectral band has been selected for pathlengthcorrection, pathlength correction terms can be determined for eachspectrum in the hyperspectral image. Relative pathlength for each pixelwas determined by assigning a relative pathlength of one to the averagespectrum of the 1440 cm⁻¹ band and using the average spectrum in a CLSprediction of all sample image spectra. The CLS analysis also included asimultaneous fit of linear baseline components to the band. Relativepathlengths varied from 0.13 to 2.5. The thinnest pathlengths were atthe edges of the sample. Because the spectra contained artifacts fromthe imaging system, the spectra were not scaled for pathlength sincethis process could serve to increase the variance of the artifact in theimage. Rather, the MCR was performed on the uncorrected spectra, and theestimated concentrations were corrected for relative pathlengthvariations.

The primary goal of the analysis was to obtain the pure-componentspectra and concentration maps of each component in the image directlyfrom the spectral data, since standards were not available for thesesamples. As described above, quantitative analysis without standards canbe accomplished with the MCR algorithm using constrained alternatingleast squares in an iterative process. Constraints were applied to limitthe range of possible solutions in the analysis and minimize thedetrimental effects of the system artifacts on the analyses. Theseconstraints comprised both non-negativity of the concentrations and thepure-component spectra, since real concentrations and spectra willconsist of all positive values, and equality constraints.

The MCR analysis requires a starting point for the pure spectra and/ortheir corresponding concentrations. In the analysis described herein,pure component spectra obtained from the SIMPLISMA algorithm were usedas starting points. These pure-component spectra are generated byfinding and extracting the purest spectral bands in the spectral datasets one at a time. From these purest pixels, often reasonable initialestimates of the pure-component spectra can be obtained. Random numberscan also be used as starting points.

Initially, MCR was performed starting with the SIMPLISMA estimatedpure-component spectra using only non-negativity constraints. Theconstrained alternating least squares algorithm used was a modificationof the fast non-negative least squares algorithm from the previouslyreferenced Bro et al. Changes to Bro's algorithm were made to speed theconvergence of the MCR analysis. The spectral data were heavilycontaminated by systematic errors, probably due to readout problems fromthe FPA detector, that severely degraded the quality of the both thepure-component spectra and the concentration maps generated from the MCRanalysis. The result is the presence of large correlated residuals inthe spectral domain and a banding artifact in the concentration maps.

The standard method of applying MCR with non-negativity constraints tospectral data can be improved by augmentation of the calibration andprediction solutions with estimates of the error covariance structure inthe spectra. All of the current implementations of the MCR algorithmsassume that the spectral errors are random, of constant variance andindependent (i.e., uncorrelated). These assumptions were violated in theFT-IR image data. The ACLS methods of the present invention enable theMCR analysis to be corrected for the presence of non-constant andcorrelated spectral errors in the data. To correct the MCR analysisusing the ACLS methods, an estimate of the error covariance structure ofthe spectral image data is required. This estimate can be obtained fromthe repeat image spectral differences of the sample. If the sample doesnot move and does not change over time, then any changes in the imagespectra at a given pixel are related to systematic changes in the systemresponse as well as random noise.

In the past, E_(A) in Eq. (1) has generally been assumed to consist ofidentically distributed uncorrelated errors of zero mean. However,instrument drift, system artifacts, and a variety of possible errorsthat occur in collecting the interferogram can generate correlated errorstructure. The conversion to absorbance also has the effect ofgenerating errors with non-uniform error variance. The error covariancestructure of E_(A) has often been estimated in remote sensingapplications with hyperspectral images with a shift difference generatedfrom a single hyperspectral image. This shift difference assumes thatthe signal between pixels is slowly varying and, therefore, the spectraldifference between adjacent pixels is representative of noise. The shiftdifference must be used if direct repeat image spectra cannot beobtained, for example in the case of a moving detector in remote sensingapplications. However, in the laboratory, repeat image spectra can beobtained, and the difference between these images can be used to obtainan estimate of the error covariance structure of E_(A). Since errors inthe image data described herein are correlated, a PCA analysis of E_(A)can serve to separate the correlated errors from the random error.

In Eq. (6), TP is the product of scores and eigenvectors from the PCA ofE_(A) for the r scores and eigenvectors that represent the significantcorrelated errors contained in E_(A). The number of eigenvectors wasselected based on a visual examination of the corresponding scoreimages. Score images with nonrandom patterns were retained. E thencontains primarily the random errors that are expected in any leastsquares analysis. To estimate C and K without the detrimental influenceof T and P that can contaminate the concentration and pure-componentspectral estimates, C can be augmented with T during calibration, or Kcan be augmented with P during prediction, or both. By augmentingcolumns of C with selected columns from T, the least squares estimate ofK is forced to ignore any pattern in the image that has the same spatialdistributions as any given set of column scores in the T vectors thatare used to augment C. By augmenting the rows of K with selected rowsfrom P, the least squares estimate of C is required to ignore anyspectral shape contained in any given row eigenvector of P vectors thatare used to augment K. The corresponding ACLS calibration and predictionequations that are part of the alternating least squares MCR procedureare then given by equations (22) and (23), respectively. In practice,the MCR method implements the augmentation by using equality constraintsto force the estimates of K and C to ignore sources of spectral andspatial variation that are contained in the augmented {tilde over({circumflex over (C)})} and {tilde over ({circumflex over (K)})}matrices.

Results From the Experimental Data

A visible video image of the approximate portion of the aged neoprenesample imaged by the FT-IR spectrometer is displayed in FIG. 12. Aslight darkening from the center to the edge of the sample is the onlyindication in the visible image of the degradation of the sample due tothe aging. The 1269 spectra from image pixels representing the sampleare presented in FIG. 13A. Note that there are significant complexbaseline variations present in these data. The spectra that weresubjected to linear baseline correction for each of the six significantspectral bands of neoprene are presented in FIG. 13B. The preprocessedspectra in FIG. 13B were subjected to the MCR analysis.

As mentioned in the Experimental section, systematic artifacts werepresent in the FT-IR spectral image data. These artifacts were notalways readily observable in the raw interferograms, single-beamspectra, or absorbance spectra. However, a PCA analysis of the spectralimage data revealed the presence of the artifact as a systematic stripedor banded pattern in the score images from the PCA analysis. Asindicated in FIG. 14, this striped pattern was apparent in the PCAscores of interferograms, single-beams, and absorbance spectra obtainedfrom the spectra of different sample or background images. The relativemagnitude of the artifact varied considerably between various image datasets as evidenced by how many eigenvectors were generated before theartifact became noticeable. The data in FIG. 14 illustrates collectedimage data where the presence of the artifact varied from very small tosignificant as indicated by the fact that the striping artifact firstbecomes visible in factor 2, 5, or 10, depending on the image. Todemonstrate the value of the ACLS methods applied to the MCR analysis, aspectral image where the artifact was a significant portion of the datawas chosen.

Not only is the artifact clearly present in the PCA score images, it isalso present in the spectral data. In FIG. 15 are shown themean-centered difference spectra between repeat images of the agedneoprene sample, in which the systematic error (noise) can be observed.Clearly, the noise in the difference spectra in FIG. 15 is not uniform.In fact, as expected by theory, the noise is higher in those regionswhere the absorbances are larger. Thus, the spectral noise is higherwhere the neoprene sample absorbs the IR beam. What is not clear fromFIG. 15 is that the noise in the sample is also correlated betweenspectral frequencies. Thus, if the error is high at frequency p, ittends to be high at surrounding frequencies. The fact that the noise ishigh in broad spectral regions is not necessarily an indication ofcorrelated error; it could simply be an indication that the noise ishigher in broad spectral regions but uncorrelated between frequencies.

The non-uniform and correlated nature of the noise in the spectralimages can best be observed by forming the error covariance matrix fromthe mean-centered repeat image spectral differences. Thistwo-dimensional representation of the spectral error in the image isshown in FIG. 16A. FIG. 16B shows the ideal noise behavior of uniformvariance and uncorrelated errors (i.e., only diagonal elements ofconstant magnitude). Clearly, the true error covariance structure of thespectral image data has non-uniform diagonal variance components andlarge off-diagonal terms, demonstrating that errors are highlycorrelated between frequencies. Thus, the errors observed from themeasured spectral image data are a far departure from the ideal behaviorpresented in FIG. 16B.

The non-constant noise variance for infrared absorbance spectra isexpected, since the log transformation required to obtain absorbancespectra takes noise variance that is expected to be constant at allfrequencies for MCT detectors and makes the noise nonuniform over thespectral frequencies. The expected form of the noise variance of thedifference of two repeat absorbance spectra at a single frequency can beestimated as outlined below. $\begin{matrix}{A = {{\log \frac{\left( {I_{s} + \sigma_{s1}} \right)}{\left( {I_{s} + \sigma_{s2}} \right)}} = {{\log \left( {I_{s} + \sigma_{s1}} \right)} - {\log \left( {I_{s} + \sigma_{s2}} \right)}}}} & (25)\end{matrix}$

where I_(s) is the noise-free single-beam intensity of the two repeatsample spectra and σ_(s1) and σ_(s2) are the standard deviations of thenoise from repeat spectra 1 and 2, respectively. From Eq. (25),estimates of the error variances in absorbance (σ_(A) ²) can be obtainedby taking a Taylor series expansion of the error variance in thesingle-beam intensities in Eq. (25) and retaining only the firstnon-zero term for both terms on the right-hand side of Eq. (25). Theresult is: $\begin{matrix}{{\sigma_{A}^{2} \approx {{\left( \frac{\delta \quad A}{\delta \quad I_{s}} \right)\sigma_{s1}^{2}} + {\left( \frac{\delta \quad A}{\delta \quad I_{s}} \right)\sigma_{s2}^{2}}}} = {{\frac{\sigma_{s1}^{2}}{I_{s}^{2}} + \frac{\sigma_{s2}^{2}}{I_{s}^{2}}} \approx \frac{2\quad \sigma_{s}^{2}}{I_{s}^{2}}}} & (26)\end{matrix}$

where the last term on the right-hand side of Eq. (26) assumes that theerror variance is comparable for both repeat single-beam spectra. FromEq. (26), the error variance for the absorbance spectra is inverselyproportional to the square of the single-beam intensity for the infraredspectrum. In a simple weighted least squares fit of the data, theweights would be proportional to the square root of the inverse of thespectral absorbance variance. If experimental estimates follow theexpected theory, then the approximate proper weights would beproportional to the average single beam spectrum obtained from the imagespectra.

FIG. 17 compares the estimated weighting function for absorbance errorvariance obtained from the repeat sample image differences and thetheoretical weighting function based on the average single-beamintensity obtained from the sample image data. Clearly, the experimentaland theoretical estimates for the weighting function are very similar,demonstrating that the repeat spectral differences can lead toreasonable estimates for the error variance of the absorbance spectraldata. Confirmation that the full covariance structure of the residual iscaptured by the repeat image spectral differences enables the use ofthis information to reduce or eliminate systematic errors during theimage analysis.

Conventional MCR methods were applied to the spectral image datapresented in FIG. 13B using non-negativity constraints on bothconcentrations and pure-component spectra. Three pure componentsappeared to be adequate to represent the original thermally modifiedneoprene and the decomposition products. Using more than three purecomponents yielded results that were not as physically meaningful asobtained with three components only. The normalized MCR pure-componentspectra obtained with three components are presented in FIG. 18. Thetrace corresponding to component 1 is characteristic of the thermallyaged but unoxidized neoprene sample. The component 2 trace indicates adecomposition product that contains a significant carbonyl decompositionproduct. The component 3 trace indicates that a second decompositionproduct is present in the aged sample that is characterized by ahydroxyl dominated decomposition product with very little formation of acarbonyl species. This second decomposition product has a differentspatial profile in the sample than the first. The high-frequency regionof the pure-component spectra is quite noisy.

Pathlength-corrected relative concentration maps representing the threepure components are presented in FIG. 19. Some of the striped artifactpresented in FIG. 14 is clearly visible in at least two of theseconcentration maps, indicating that the MCR analyses are corrupted bythis artifact. The carbonyl decomposition product (component 2 in FIG.19) demonstrates a large concentration gradient that is high at theouter edges of the sample. The hydroxyl component decomposition productshown as component 3 in FIG. 19 is more variable and extends somewhatbeyond the region containing the carbonyl decomposition product. Thethermally aged but unoxidized neoprene shown as component 1 in FIG. 19is present somewhat uniformly throughout the sample although it may havebeen reduced near the edge of the sample by the oxidation.

MCR was then applied to the same spectral image data with theconcentration matrix augmented by the first five sets of scores from themean-centered repeat image spectral differences, according to the ACLSmethod of the present invention. The score-augmented MCR was implementedwith equality constraints on these five sets of image scores. Inapplying these equality constraints on the scores, the alternatingclassical least-squares generation of the pure-component spectra iscorrected for the presence of score-related concentration errors in theimage data. FIG. 20 presents the three pure-component spectra generatedfrom the MCR augmented with the scores. Note that the noise in thehigh-energy region of the pure-component spectra is greatly reduced inthese pure-component estimates as compared to FIG. 18. In addition, moredetailed structure becomes available in the OH stretching region of thespectra (3000-3700 cm⁻¹ spectral region) with score augmentation. Thederivative nature of the hydroxyl band for the carbonyl dominateddecomposition product (component 2) may indicate some hydrogen bondingwith the hydroxyl dominated decomposition product. Since theconcentrations were not corrected when augmenting with scores only, theconcentration maps indicate essentially no improvement in the stripingartifact relative to the standard non-augmented MCR analysis of the samedata.

MCR was then applied to the same spectral image data with the first fivesets of eigenvectors from the mean-centered repeat image spectraldifferences added to the pure-component spectral matrix. Theeigenvector-augmented MCR was implemented with equality constraints onthese five eigenvectors. With eigenvector augmentation only, there wassome improvement in noise of the high-energy side of thehydroxyl-dominated pure component, but there was little, if any,improvement in the noise on the other pure-component spectra. However,the concentration maps exhibit dramatic improvement in the quality. Thestriping artifact was nearly completely removed when the eigenvectorsaugment the MCR, as shown in FIG. 21. These improvements inconcentration maps were expected since the augmentation of thepure-component spectra with the eigenvectors corrects the concentrationestimates.

Finally, the MCR analysis was augmented with both scores andeigenvectors from the repeat image spectral differences. In thisembodiment of the present invention, one simply augments with scoresduring the calibration phase of the MCR and augments with theeigenvectors during the prediction phase. Thus, at each iteration in theMCR procedure, any estimates related to the augmented vectors arereplaced with the original eigenvectors from the PCA decomposition ofthe repeat image differences. The combined augmentation with scores andeigenvectors generates both improved pure-component spectra andcorrected concentration maps. The pure-component spectra with scores andeigenvector augmentation are presented in FIG. 22. These spectra havethe low noise of the scores-augmented MCR and are also slightlymodified, especially in the high-energy hydroxyl spectral region. Theconcentration maps with scores- and eigenvector-augmented MCR arepresented in FIG. 23. Like the eigenvector-augmented MCR concentrationmaps, these images exhibit greatly reduced striping artifact. Thereduction in the artifact is emphasized in FIG. 24, which shows thedifference between the standard MCR and the fully augmented MCRconcentration maps for all three components (i.e., the differencebetween the concentration maps in FIG. 23 and FIG. 19). As indicated bythis difference map, a significant reduction in the system artifact wasachieved when augmenting with estimates of the error covarianceinformation derived from the difference between two repeat images.

Having thus described the present invention with particularity anddemonstrated its utility with respect to both simulated and actualexperimental data, those skilled in the art will appreciate that thepresent invention has broad applicability to a wide range ofopportunities for analyzing multivariate spectral data and obtainingboth qualitative and quantitative results. Many preprocessing proceduressuch as centering, scaling, pathlength correction, smoothing,derivatives, multiplicative signal correction, PCA, wavelet compression,and covariance filtering have been developed that can be performed onthe measured spectra when using the present invention.

We claim:
 1. A method for analyzing multivariate spectral data,comprising the steps of: a) creating a calibration model for acalibration set of multivariate spectral data A by: i) obtaining a setof reference component values C representative of at least one of thespectrally active components in the calibration set of multivariatespectral data A, ii) estimating pure-component spectra {circumflex over(K)} for the at least one of the spectrally active components accordingto {circumflex over (K)}=(C^(T)C)⁻¹C^(T)A=C⁺A, iii) obtaining spectralresiduals E_(A) according to E_(A)=A−C{circumflex over (K)}, and iv)augmenting the estimated pure-component spectra {circumflex over (K)}with at least one vector of the spectral residuals E_(A) to obtainaugmented pure-component spectra {tilde over ({circumflex over (K)})};and b) predicting a set of component values {tilde over ({circumflexover (C)})} for a prediction set of multivariate spectral data A_(P) by:i) further augmenting the augmented pure-component spectra {tilde over({circumflex over (K)})} with at least one vector representing aspectral shape that is representative of at least one additional sourceof spectral variation in the prediction set, and ii) predicting the setof component values {tilde over ({circumflex over (C)})} using thefurther augmented pure-component spectra {tilde over ({circumflex over(K)})} according to {tilde over ({circumflex over (C)})}=A_(P){tildeover ({circumflex over (K)})}^(T)({tilde over ({circumflex over(K)})}{tilde over ({circumflex over (K)})}^(T))⁻¹=A_(P)({tilde over({circumflex over (K)})}^(T))⁺.
 2. The method of claim 1, whereinaugmenting step a)iv) is repeated until all sources of spectralvariation are accounted for in the calibration set.
 3. The method ofclaim 1, wherein an F-test is used to select the number of vectors usedto augment the estimated pure-component spectra {circumflex over (K)}.4. The method of claim 1, further comprising the step of augmenting theestimated pure-component spectra {circumflex over (K)} with at least onevector representing at least one spectral shape representative of atleast one additional sources of spectral variation in the calibrationset prior to step a)iii).
 5. The method of claim 1, further comprisingthe step of augmenting {circumflex over (K)} to account for baselinevariations in the calibration set prior to step a) iii).
 6. The methodof claim 1, further comprising the step of augmenting {tilde over({circumflex over (K)})} to account for baseline variations in thecalibration set prior to step b).
 7. The method of claim 1, wherein thereference component values C comprise concentrations of the spectrallyactive components.
 8. The method of claim 1, wherein the spectrallyactive components are selected from the group consisting of chemicalspecie, chemical interactions among species, physical variations,temperature variations, humidity variations, spectrometer drift, changesin spectrometers, and insertion effects.
 9. The method of claim 1,wherein the spectral residuals E_(A) are obtained from a full CLS modelor a cross-validated CLS model.
 10. The method of claim 1, wherein thespectral residuals E_(A) are decomposed according to E_(A)=TP+E, where Tis a set of n×r scores and P is a set of r×p loading vectors obtainedfrom factor analysis of the spectral residuals E_(A), and E is a set ofn×p random errors and spectral variations not useful for prediction. 11.The method of claim 10, wherein the P loading vectors comprise the atleast one vector of the spectral residuals E_(A) used to augment theestimated pure-component spectra {circumflex over (K)} in step a)iv).12. The method of claim 10, wherein the factor analysis method comprisesprincipal components analysis.
 13. A method for analyzing multivariatespectral data, comprising the steps of: a) creating a calibration modelfor a calibration set of multivariate spectral data A by: i) obtaining aset of reference component values C representative of at least one ofthe spectrally active components in the calibration set of multivariatespectral data A, ii) estimating pure-component spectra {circumflex over(K)} for the at least one of the spectrally active components accordingto {circumflex over (K)}=(C^(T)C)⁻¹C^(T)A=C⁺A, iii) obtaining spectralresiduals E_(A) according to E_(A)=A−C{circumflex over (K)}, iv)decomposing the spectral residuals E_(A) according to E_(A)=TP+E where Tis a set of n×r scores and P is a set of r×p loading vectors obtainedfrom factor analysis of the spectral residuals E_(A), and E is a set ofn×p random errors and spectral variations not useful for prediction, v)augmenting the set of reference component values C with at least onevector of the T scores to obtain a set of augmented component values{tilde over (C)}, and vi) estimating augmented pure-component spectra{tilde over ({circumflex over (K)})} according to {tilde over({circumflex over (K)})}=({tilde over (C)}^(T){tilde over (C)})⁻¹{tildeover (C)}^(T)A≈{tilde over (C)}⁺A; and b) predicting a set of componentvalues {tilde over ({circumflex over (C)})} for a prediction set ofmultivariate spectral data A_(P) according to {tilde over ({circumflexover (C)})}=A_(P){tilde over ({circumflex over (K)})}^(T)({tilde over({circumflex over (K)})}{tilde over ({circumflex over(K)})}^(T))⁻¹=A_(P)({tilde over ({circumflex over (K)})}^(T))⁺.
 14. Themethod of claim 13, wherein the augmenting step a)iv) is repeated untilall sources of spectral variation are accounted for in the calibrationset.
 15. The method of claim 13, wherein an F-test is used to select thenumber of vectors used to augment the estimated pure-component spectra{circumflex over (K)}.
 16. The method of claim 13, further comprisingthe step of augmenting the estimated pure-component spectra {circumflexover (K)} with at least one vector representing a spectral shaperepresentative of at least one additional source of spectral variationin the calibration set prior to step a)iii).
 17. The method of claim 13,further comprising the step of augmenting {circumflex over (K)} toaccount for baseline variations in the calibration set prior to stepa)iii).
 18. The method of claim 13, further comprising the step ofaugmenting the augmented pure-component spectra {tilde over ({circumflexover (K)})} with at least one vector representing a spectral shaperepresentative of at least one additional source of spectral variationin the prediction set prior to step b).
 19. The method of claim 13,further comprising the step of augmenting {tilde over ({circumflex over(K)})} to account for baseline variations in the calibration set priorto step b).
 20. The method of claim 13, wherein the reference componentvalues C comprise concentrations of the spectrally active components.21. The method of claim 13, wherein the spectrally active components areselected from the group consisting of chemical specie, chemicalinteractions among species, physical variations, temperature variations,humidity variations, spectrometer drift, changes in spectrometers, andinsertion effects.
 22. The method of claim 13, wherein the step a)iv)comprises augmenting the set of reference component values C with a setof random numbers.
 23. The method of claim 13, wherein the spectralresiduals E_(A) are obtained from a full CLS model or a cross-validatedCLS model.
 24. The method of claim 13, wherein the factor analysismethod comprises principal components analysis.